User: Anna Barsukova, a graduate student in the lab of Michael Forte, a cell biologist at Oregon Health and Science University in Portland.
Project: Imaging calcium flux in mitochondria of primary neurons in response to cellular stress, a key apoptotic mechanism in neurodegenerative disease.
Problem: Barsukova needed a fast system (20 to 100 msec timeframe) with superior spatial resolution for imaging a subcellular structure. She also needed to switch easily between different light filters to track two fluorescent probes simultaneously.
Solution: Unlike confocal and two-photon microscopes, which direct light to a localized area within a focal plane, wide-field systems bathe the entire sample in light, and work well for imaging a sample about 10 µm thick. Because she studies a single layer of cultured cells, Barsukova could not justify the expense of a confocal or a two-photon microscope, which would be more suited to 3D reconstruction in the range of 50 to 300 µm. Additionally, those techniques do not have the flexibility required for manipulating filters. So she opted for a wide-field system, the Olympus IX-81 inverted microscope.
Barsukova opted for several custom features: a galvanometer for boosting the speed of probe excitation to timescale of the calcium signal, a special 150x magnification lens, a dual-view device that splits the camera chip to simultaneously view two filter modes, a computer with a terabyte of RAM, and a Z-stage and deconvolution software for better resolution and basic 3D imaging. (See "Rendering images in 3D," The Scientist, 18(8):40, 2004.) Because her 3D reconstructions are just for "pretty pictures" and not quantitative analysis, she says, she chose the software that came with the microscope rather than spending more on a better-quality package.
Wide-field systems "are sort of like a car," says Maddox: You can buy just a barebones model, or you can mix and match with extra features to tailor the system to the specifics of your experiment. "If you can build two light microscopes and optimize each of those for the same price as one confocal, people are starting to think, 'We can do more with that,'" says Ian Harper, who directs the microimaging facility in the biomedical sciences department at Monash University in Clayton, Australia.